行政院國家科學委員會專題研究計畫 成果報告
視網膜色素上皮細胞轉殖α6β4integrin 以增進黏著於細
胞外間質─視網膜色素細移殖之前驅實驗
研究成果報告(精簡版)
計 畫 類 別 ： 個別型 計 畫 編 號 ： NSC 95-2314-B-002-159- 執 行 期 間 ： 95 年 08 月 01 日至 96 年 07 月 31 日 執 行 單 位 ： 國立臺灣大學醫學院眼科 計 畫 主 持 人 ： 楊中美 共 同 主 持 人 ： 楊長豪 計畫參與人員： 碩士級-專任助理：張鳳書 處 理 方 式 ： 本計畫可公開查詢中 華 民 國 96 年 10 月 26 日
Overexpression of
and Integrin Enhances Human Retinal
Pigment Epithelial Cells Adhesion and Proliferation on Layers of
Br
uc
h’
s
Me
mbr
ane
Chung-May Yang1, I-Mo Fang1, 2, Chang-Hao Yang1, Muh-Shy Chen1
From the1Department of Ophthalmology, National Taiwan University Hospital,
Taipei, Taiwan, and the2Department of Ophthalmology, Taipei City Hospital
Zhongxiao Branch, Taipei, Taiwan
Reprint requests to:
Chang-Hao Yang, MD, PhD
Department of Ophthalmology
National Taiwan University Hospital
No.7, Chung-Shan S. Rd. Taipei, Taiwan
TEL: 886-2-23123456 ext. 3193
Fax: 886-2-23412875
Abstract.
Purpose. To elucidate the roles of integrinon retinal pigment epithelial (RPE) cells adhesion and proliferation on extracellular matrix and to investigate whether
overexpression of integrinandcould promote adhesion and proliferation of RPE cells on Bruch’s membrane.
Mehtods. The expression of integrinmRNA and surface protein in ARPE-19 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and
flow cytometry, respectively. Transfectants containing mutatedand
cDNAwhich were generated by site-directed mutagenesis, was used to test the adhesion on extracellular matrix. Mechanical and enzymatic techniques were used to
expose varied layers of Bruch’s membrane. The attachment of ARPE-19 cells
transfected with- and-cDNA on different layers of Bruch’s membrane was determined by adhesion analysis. Cell morphology and surface coverage were
evaluated by scanning electron microscopy.
Result. Integrin6and4mRNA and proteins were constitutionally expressed in
ARPE-19 cells. The adhesion rate of cells to laminin was decreased by cells
expressingmutant ormtant. The-cDNA transfectants displayed enhanced expression of the integrinandand increased adhesion to laminin, fibronectin and collagen type IV, whereas-cDNA transfectants showed increased expression of
theintegrin and enhanced attachment only to laminin. In Bruch’s membrane explant model,transfectants promoted cell attachment and proliferation on all
layers of Bruch’s membrane, whereastransfectants enhanced adhesion and
proliferation on basal lamina and external collagenous layers.
Conclusions. Overexpression ofandintegrin could enhance ARPE-19 cells adhesion and proliferation on Bruch’smembraneexplant.Modification ofintegrin expression in RPE cells to promote cellular adhesion and proliferation might be a
Introduction
Aged-related macular degeneration (AMD) is the leading cause of visual impairment
in developed countries in patients over 50 years of age.1, 2Currently, there is no
satisfactory treatment to reverse the visual loss in AMD.3-5Excision of submacular
choroidal new vessels (CNV) combined with retinal pigment epithelial (RPE) cells
transplantation that provides the chance to remove pathological lesions and prevent
secondary choriocapillaris atrophy, has been proposed as a treatment for choroidal
neovascularization.6-8However, little patients gain significant visual improvement
from such surgery9, 10Increasing evidences suggest the inability of transplanted RPE
cells to attach and survive on Bruch’s membrane (BM) is one of the major causes for
RPE transplant failure and poor visual outcomes.11, 12Therefore, to enhance adherence
and repopulation of transplanted RPE cells to Bruch’s membrane became the key step
for the success of RPE transplantation.
RPE transplantation is often performed after surgical removal of subfoveal
neovascualr membranes, which can damage and expose deeper layers of BM, such as
inner collagenous layer, elastic layer and even outer collagenous layer.13, 14Thus, RPE
cells harvested for transplantation usually need to adhere, proliferate and function on
deeper layers of BM. The anatomic layer of BM available for RPE attachment affects
of the attached cells.15Several in vitro studies demonstrated that the RPE
reattachment rate was highest to the inner aspects of BM and decreased as deeper
layers of BM are exposed.15, 16Zarbin had reported that freshly harvested aged human
RPE cells fail to attach onto the basal lamina or ICL of BM, because they do not
express integrins necessary for attachment.17He found that up-regulation of integrins
by culturing promotes more efficient RPE attachment to and survival on BM. This
finding suggested that selective up-regulation of integrins responsible for adhesion to
BM is a promising strategy to enhance transplanted cells to attach to host BM.
The attachment of RPE cells to Bruch’s membrane is mainly mediated by an
interaction between of integrins on the RPE surface and ligands in the extracellular
matrix, including laminin, fibronectin, vitreonectin and collagen.18, 19Integrins are
heterodimeric molecules composed of a noncovalently boundandsubunit.20-22 There are 18and 8subunits that are known to assemble into 24 distinct integrins.23,
24
. Integrinis a receptor of laminin, the major component of Bruch’s membrane.
25, 26
Integrinis widespread expressed at the basal surface of most epithelial cells and plays an essential role in cell adhesion to the underlying basement membrane.27,
28
.Additionally, integrinalso contributed to proliferation, migration and differentiation of cells.29, 30Previous electron microscopic (EM) studies of the
junction of RPE and BM revealed that hemidesmosomes was present in the basal
surface of RPE cells, adding in maintaining cohesion between RPE and Bruch’s
membrane.31, 32Since hemidesmosome is mainly composed of integrinthis finding implied that integrinmay also present on the surface of RPE cells. However, the roles of integrinon RPE cells adhesion to extracellular matrix have not been investigated.
In this study, we investigated the expressions and the roles of integrinin
ARPE-19 cells attachment to extracellular matrix. Furthermore, we determined
whether genetically modified RPE cell to overexpress integrinandsubunit could enhance adhesion and proliferation on various layers of Bruch’s membrane.
Materials and Methods
Antibodies and reagents
Monoclonal antibodies used in this study were: anti-integrin6(clone GoH3),
anti-integrinclone 3E1) and anti-integrinclone 6S6) from Chemicon
(Temecula, CA). Laminin, fibronectin, vitreonectin and collagen type I were obtained
from Sigma-Aldrich (St. Louis, MO).
Cell culture
ARPE-19 cells were purchased from the Bioresource Collection and Research Center
(BCRC, Hsinchu, Taiwan) and cultured in Dulbecco’smodified Eagle’smedium/F-12
human aminiotic membrane nutrient mixture (DMEM/F-12; Sigma-Aldrich, St. Louis,
MO) with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) in a humidified
incubator at 370C in an atomosphere of 5% CO2.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from ARPE-19 cells with Trizol reagent (Invitrogen-Gibco, Grand Island,NY,USA)according to themanufacturer’sinstructions.Onemicrogram of total RNA from each sample was annealed for 5 min at 65C with 300ng
U Moloney murine leukemia virus reverse transcriptase (MMLV-RT)
(Invitrogen-Gibco, Grand Island, NY, USA) per 50g reaction for 1 h at 37C. The reaction was stopped by heating for 5 min at 90C.
The cDNA obtained from 0.5 μg totalRNA wasused asatemplateforPCR
amplification. Oligonucleotide primers were designed based on Genbank entries for
human,and β-actin. The following primers were used for amplification
reaction: For, forward primer 5'- GTGTTGCCAACCAGAATGGCTCGC-3′; reverse primer 5'-CAGTCACTCGAACCTGAGTGCCTGC-3’; For, forward primer5′-ACAGGAGGGGTTAAAGCTGC-3′;reverseprimer5′
-GCAGCTTTAACCCCTCCTGT-3′;Forforward primer
5'-CCTCATACTTCGGATTGACC; reverse primer 5’
-TGTTCAGTGCAGAACCTTCA-3; For β-actin,forward primer5′-GAAC
CCTAAGGCCAACCGTG-3′;reverseprimer5′-TGGCATAGAGGTCTTTA CGG-3′.
The amplification was performed in 30 cycles at 55 °C, 30 s; 72 °C, 1 min; 94 °C,
30 s. PCR products were separated by performing gel electrophoresis on 2% agarose
containing ethidium bromide (Sigma, St. Louis, MO, USA) and then analyzed under
ultraviolet light against the DNA molecular length markers. The intensity of the
Kodak, Rochester, NY, USA), and the amount of PCR-amplifiable material in each
reverse-transcribed sample was standardized against the amount of a housekeeping
gene-actin.
Plasmids and Generation of Point Mutation by Site-directed mutagenesis
Plasmids containing the full-lengthintegrin cDNA (pWT) andintegrin cDNA (p4WT) were gift from Dr. Giancotti. Plasmid p6mut, encoding the mutated S47L
integrin, was generated by polymerase chain reaction (PCR) amplification using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), primers
(forward) 5’-AGCCTCTTCGGCTTCTTGCTGGCCATGCAC-3’and (reverse)
5’-GCCAGTGCATGGCCAGCAAGAAGCCGAAGA-3’and plasmid pWT as a template; the primer sequences of plasmid4mutext,encoding the mutated Q155L4
integrin, were: (forward)5’-TCAGCGTCCCGCTGACGGACAT GAGGC-3’and
(reverse)5’-TCATGTCCGTCAGCGGGACGCTGA-3’and plasmid p4WT as a
template; the primer sequences of plasmid4mutint, encoding the mutated R1281W
4integrin, were: (forward)5’-AACCCTAAGAACTGGATCGTTG CTTATTG-3’and
(reverse)5’-CAATAAGCAGCATCCAGTTCTTAGGGTT-3’and plasmid p4WT as
Transient Transfections
ARPE-19 Cells were seeded in 6-well tissue culture plates at 1.3x 105cells per well
and growth for 48 h in MEM/10% FCS at which time they reach about 90%
confluence. The medium was replaced with serum-free medium, and after 4 hours of incubation the cultures were transfected with 2g of each mutanted construct or full-length integrin6or4cDNA constructs or corresponding empty constructs,
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA)in accordance with the
manufacturer’s instructions. Transfection efficiency was analyzed 24 hours after
transfection by determining the population of cells with GFP expression.
Flow cytometry
Subconfluent cells were washed twice with phosphate-buffer saline (PBS) and
harvested by trypsin/ethylenediamine tetraacetic acid (EDTA) (0.25%wt:vol, 5mM).
Cells were washed once in PBS containing 10% FBS. Cells were incubated with
monoclonal anti-integrin antibodies for 60 minutes at 40C and washed twice with PBS.
Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-rat6 antibody (Jackson
ImmunoResearch laboratories, West Grove, PA, USA) or phycoerythrin
(PE)-conjugated anti-mouse4 secondary antibody (Sigma-Aldrich, St. Louis, MO)
resuspended in 0.5ml PBS with 10% FBS. Labelled cells were scanned on a
FACSCalibur cytometry (Becton Dickinson) and analysed using CellQuest software.
For each sample the data from 1x104events were collected. All data are presented as
the mean fluorescence intensity, which is proportion to the logarithm of the
fluorescence intensity.
Preparation of different layers of BM
Bruch’s membrane explants were prepared as previously described by Del Priore and
Tezel32. In brief, a full-thickness circumferential incision was made posterior to the
ora serrata and the anterior segment and vitreous were carefully removed. Four radial
incisions were then made and the sclera was peeled away. A circumferential incision
was made into the subretinal space 1 mm posterior to the ora serrata. The
choroids-BM-RPE complex was then carefully peeled toward the optic disc and
removed after trimming its attachment to the optic nerve. Native RPE was removed
by bathing the explant with 0.02N ammonium hydroxide in a 50-mm polystyrene
petri dish (Facon, Becton Dickinson, Lincoln Park, NJ) for 20 minutes at room
temperature, followed by washing 3 times in phosphate-buffered saline (PBS).
polycarbonate-polyvinylpyrrolidone membrane with 0.4-m pores (Millipore, Bedford, Mass) with the basal lamina facing toward the membrane. The agarose was allowed to solidify at
40C, and the hydrophilic membrane was peeled off along with the basal lamina (BL)
of the RPE, thus exposing the bare internal collagenous layer (ICL). Triplicate buttons
from each eye were further treated at 370C with1-mg/ml collagenase (Sigma-Aldrich,
St. Louis, MO) in PBS at pH 7.5 for 1 hour to remove the internal collagenous layer
and expose the elastic layer (EL) and with collagenase followed with 20 U/ml elastase
(Sigma-Aldrich, St. Louis, MO) in PBS at pH 8.5 for 1 hour to digest the internal
collagenous and elastin layers and expose the external collagenous layer (ECL). After
the enzymatic treatment, 6 mm diameter peripheral buttons were trephined and placed
on 4% agarose in untreated polystyrene 96- well plates. The well were gently rinsed
with PBS 3 times for 5 minutes and then stored at 40C.
RPE reattachment Studies
Plates were pre-coated with the ECM molecules: laminin (10g/ml), fibronectin ( 10 g/ml), vitronectin (10 g/ml ) or collage type IV (25g/ml) in PBS, in a total volume of 100L, for 4 hours at room temperature. Fifteen thousand RPE cells were plated
on 96-well tissue culture plates pre-coating with ECM or different layers of Bruch’s
penicillin G, 100g/ml streptomycin, 5 g/ml gentamicin, and 2.5 g/ml amphotericin B was added to reach a final volume of 200L in each well. Cells were allowed to
attach to the surface of ECM for 6 hours or Bruch’s membrane for 24 hours in a
humidified atmosphere of 95% air and 5% carbon dioxide at 370C in phenol red-free
MEM. Unattached cells were removed from the tissue culture plates by gently
washing the wells 3 times with MEM
Assay for RPE adhesion
The number of attached live RPE cells in each well was determined with a
colorimetric assay that indirectly estimates the number of live cells by measuring
intracellular dehydrogenase activity (CellTiter 96 Aqueous one solution cell
proliferation assay; Promega, Madison, WI). The dehydrogenase enzymes in live cells
reduce MTS (3-(4,5-dimethyothiazol-2-yl)-5-(3-carboxymethoxyphenyl)
-2-(4-sulfophenyl)-2H-tetrazolium) into formazan in the presence of phenazine
methosulfate (PMS). The quantity of the formazan product can be determined from
the absorbance at 490 nm is directly proportional to the number of living cells in
Twenty microliters of freshly prepared MTS/ PMS solution (20:1) was added to each
well, resulting in a final concentration of 333 µg/mL of MTS and 25µmol of PMS.
Plates were incubated for 4 hours at 37°C; 100 µL of medium from each well was
transferred to the corresponding wells of another 96-well plate and read at 490nm
using an enzyme-linked immunosorbent assay plate reader. The corrected absorbance
was obtained by subtracting the average optical density reading from triplicate sets of
control sets containing the BM explant on 4% agarose without plated cells. The RPE
adhesion ratio was as follows: cell adhesion (%) = (mean absorbance of cell attached
to wells/ mean absorbance of total number of cells plated) x100. Pilot studies were
performed to verify that there is a linear and reproducible relationship between cell
number and absorbance at 490nm (data not shown).
Assay for RPE Proliferation
Fifteen thousand ARPE-19 cells were planted on different layers of BM as described
above. Cells were maintained for 24 hours in serum-free MEM containing 100 IU/mL penicillin G, 100g/ml streptomycin, 5 g/ml gentamicin, and 2.5 g/ml amphotericin B. At the end of this period, cell proliferation was stimulated by replacing the medium
with MEM supplemented with 15% fetal bovine serum (FBS) and 1 ng/ml
MTS assay 24 hours after growth stimulation as described above. The proliferation
ratio was the ratio of the number of viable and attached cells 24 hours after growth
stimulation to the initial number of viable and attached cells on a certain explant.
Scanning electron microscopic analysis
Fifteen thousand RPE cells were plated on different layers of Bruch’s membrane
explants, and serum- and phenol-free MEM containing 100 IU/mL penicillin G, 100g/ml streptomycin, 5 g/ml gentamicin, and 2.5 g/ml amphotericin B was added to reach a final volume of 200L in each well. Cells were allowed to attach to
and proliferate on the surface of different layers of Bruch’s membrane for 7 days in a
humidified atmosphere of 95% air and 5% carbon dioxide at 370C. After gently
washing the wells 3 times with MEM, Bruch’s membrane explants were fixed in
modified Karnovsky fixative (2.5% glutaraldehyde and 2% paraformaldehyde in
0.1M cacodylate buffer [pH 7.4]) at 40C overnight. They were then postfixed in 1%
osmium tetroxide in 0.16 M cacodylate buffer (pH 7.4) for 1 hour, stained in 1%
uranyl acetate buffer, and dehydrated in a graded series of ethyl alcohol (30%-100%).
The samples were then critical point dried ( E 3000; Polaron, Watford Hertfordshire,
UK), mounted on aluminum specimen stubs with carbon-conductive tabs grounded
Polaron). Samples were examined by SEM (model S-4500 FEG; Hitachi, Tokyo,
Japan) at 15kV accelerating voltage and the images recorded (55P/N film; Polaroid
Corp., Cambridge, MA)
Statistical analysis
Student’st-test was used to compare data between two groups. To compare data among three or more groups, one-way analysis of variance (ANOVA) followed by the
Bonferroni test was used. Data are expressed as means ± SEM and P < 0.05 was
Results:
Expression ofintegrin6 and subunit mRNA as well as protein in ARPE-19 cells
Integrinsubunit and4subunit mRNAs were detectable in ARPE-19 cells (Fig 1A).
Flow cytometric analysis revealed that integrinsubunit and4 subunit proteins
were also presented on the surface of ARPE-19 cells (Fig 1B).
Transient expression of mutant6andin ARPE-19 cells and their effects on
cells adhesion to laminin
To investigate the role of integrin6in RPE cell adhesion to extracellular matrix,
we selectively mutated the amino acid residues of integrinand4, which are
critical for binding function, and then tested the binding ability to laminin. Three
cDNA constructs: each contained single amino acid mutant:mutant by S→L substitution at position Serine 47(mut);mutant by Q→L substitution at position glutamine155 (mutex) andmutant by R→W substitution at position arginine
1281(mutin) were generated by site-directed mutagenesis. Sequencing of the
products confirmed that only the intended mutation was introduced (data not shown).
surface expression of integrin6subunit andsubunit were determined by flow
cytometry. The expression of integrinsubunit andsubunit in mock-transfectants was similar to that in parental cells. Transfection with themut cDNA construct led to a significant decrease in surface expression of integrinsubunit, but had no effects onsubunit, compared to parental cells. Similarity,mutex-transfectants
exhibited a significant decrease in the expression of integrinsubunit, but no difference in surface expression of integrinsubunit compared to parental cells. However, there was no difference in surface expression of both6andsubunit
betweenmutint-transfectants, and parental cells (Figure 2A).
We next examined the adhesion ofmut-,mutex-,mutin-transfectants,
mock-transfectants and parental cells to laminin. Transfection with themut cDNA andmutexcDNA resulted in a statistically significant reduction in binding to
laminin, compared with mock- transfectants and parental cells, whereas
mutin-transfectants did not display significant differences in ligand binding to
laminin than mock-transfectant and parental cells (Fig 2B).
Effects ofcDNA -transfectants,cDNA -transfectants on adhesion of cells to
To study whether transfection ofcDNAandcDNAconstruct into ARPE-19
cell could enhance binding to various extracellualr matrixes, we first examined the
expression levels of,and4subunits on the surface ofcDNA-and
cDNAtransfectants by flow cytometry analysis. As shown in Figure 3A,
transfection with thecDNA construct into ARPE-19 cells resulted in overexpression of the integrin6,andsubunit on cell surfaceHowever,
transfection with thecDNAconstruct led to the overepxression of both6 and subunits in the surface of ARPE-19 cells. The level of1 subunit appeared no change incDNAtransfectants.
Next, we performed adhesion assays to determine the adhesion of
cDNAtransfectants andcDNAtransfectants tothe extracellular matrix: laminin (LN), fibronectin (FN), vitreonectin (VN) and collagen type IV (Col IV). The cDNA -transfectants showed a significant increase (P < 0.05) in adhesion to LN and FN compared to mock-transfectants and parental cells. No statistically significant
difference was found in binding ability to Col IV and VN amongcDNA -transfectants, mock-transfectants, and parental cells. In addition,
to mock-transfectants and parental cells. There were no statistically significant
differences in binding ability to FN, VN and Col IV amongcDNA -transfectants , mock-transfectants, and parental cells (Figure 3B).
Effects ofcDNA -transfectants,cDNA -transfectants on adhesion of cells to
different layers of Bruch’s membrane
To study whether transfection ofcDNAandcDNAconstructs into ARPE-19 cells could enhance adhesion to possible planes of BM after submacular surgery, we
compared the adhesion betweencDNA -transfectants,cDNA -transfectants and mock-transfectants, parental cells to different layers of Bruch’s membrane. The cDNAtransfectants showed a significant increase (P < 0.05) in adhesion to BL, ICL, EL and ECL of BM compared to mock-transfectants and parental cells. In
contrast, thecDNAtransfectants showed a significant increase (P < 0.05) in adhesion to BL and ICL than mock-transfectants and parental cells, but not to EL and
ECL (Figure 4A).
Effects ofcDNA -transfectants,cDNA -transfectants on proliferation rates
The proliferation rates ofcDNAtransfectants on BL, ICL, EL and ECL of BM were significantly higher than those of mock-transfectants and parental cells. The cDNAtransfectants showed significantly increased proliferation (P < 0.05) on BL, ICL and EL than mock-transfectants and parental cells (Figure 4B).
The ability ofcDNA- andcDNA- transfectants to repopulate different
layers of Bruch’s membrane
To investigate whether transfection with thecDNAandcDNA constructs into ARPE-19 cells could promote proliferation and repopulation to various layers of
Bruch’s membrane, we compared the surface coverage and morphology of
transplanted cells betweencDNA-,cDNA- and mock-transfectants to different layers of Bruch’s membrane after an incubation period of 7 days by scanning electron
microscope (SEM). ThecDNA-transfectants could almost completely cover the explant with BL, ICL, EL and ECL. SEM ofcDNA-transfectants on BL had formed a continuous layer with fairly uniform hexagonal shape (Figure 5A). On ICL, cDNA-transfectants were also flattened and formed a continuous layer, but some gaps and small defects in the monolayer are present (Figure 5B). SEM ofcDNA
-transfectants on EL showed many defects in Bruch’s membrane coverage.
Resurfacing RPE cells tended to be of variable morphology and flattened. On the
margin of defects, numerous pseudopodia extended from cells to the defects (Figure
5C). On ECL, despite the explants were almost complete coverage, many large RPE
defects were present. Some cells remained round without flattened in the margin of
defects (Figure 5D).
ThecDNA -transfectants could resurface the explants with BL, ICL. Similar to cDNA-transfectants,cDNA-transfectants on BL could form a continuous layer with fairly uniform hexagonal shape (Figure 5E). SEM ofcDNA-transfectants on ICL showed almost complete coverage by flat cells, but many RPE defects several
cell diameters wide were present (Figure 5F). ThecDNA-transfectants could not survival on EL and ECL. SEM showed that no intact cells were present, revealing the
underlying elastic and collagen fiber of Bruch’s membrane bed (Figure 5G, H)
SEM of mock-transfectants on BL showed complete coverage, but some small defects
were present (Figure 5I). However, mock-transfectants could not be survival on ICL,
0Discussion
RPE cells that remain unattached to a substance after delivery into the subretinal
space may undergo apoptosis, so it is important to optimize the conditions for RPE
reattachment to Bruch’s membrane in order to promote graft survival.35In this study,
we demonstrated that6and4integrin subunits were present on the surface of
ARPE-19 cells, mediating the adhesion function to laminin, the major components of
Bruch’s membrane. Transduction of integrin6orgene into ARPE-19 cells
resulted in overexpression of both integrin6and4on cell surface, with significantly
increased adhesion to laminin. In Bruch’s membrane explant model,and –transfectants also exhibit better adhesion and proliferation on deeper layers of
Bruch’s membrane than mock-transfectants or parental cells. Taken together, these
results indicated that genetically modified ARPE-19 cells that overexpression of integrin could enhance cells adhesion and proliferate on deeper layers of Bruch’s membrane explant. Modification of integrin expression in RPE cells to
promote cellular adhesion and proliferation might be an alternative strategy to
increase the successful rate of RPE transplantation.
In human, pathogenic mutations in either theor thegenes result in blistering
with pyloric atresia, in which expression of theorintegrin is aberrant.36, 37In transgenic mouse studies, ablation oforintegrin subunits produced a phenotype of severe mucocutaneous fragility, phenotypically similar to human diseases.38These
findings emphasized the importance of theintegrin in the adhesion of epithelial cells to the basement membrane.
Several experimental studies had demonstrated the potential of transplantation into
subretinal space of RPE cells genetically modified to produce desired factors to treat
retinal degenerative diseases such as AMD.39Saigo et al., showed that RPE cells
transduced with brain derived-neurotrophic factor (BDNF) gained the ability to
express this factor in subretinal space without induction of host immunologic
reaction.40Lund et al., showed that transplantation of genetically modified RPE cells
to extend their in vitro lifespan into the subretinal space could support photoreceptors
survival and limit the deterioration of visual function in RCS rats.41In the present
study, we widen the scope of ex vivo gene transfer to RPE cells in adjunctive
molecular treatments for AMD by demonstrating the feasibility of genetically
modified ARPE-19 cells to overexpressintegrin in enhancing cells adhesion and
Specificity-determining loops (SDL) segment in the I-like domain ofintegrinand propeller domain of integrin were found to play crucial roles in the function of ligand binding.42, 43Tsuruta and coworkers demonstrated that substitution of
glutamine residue for leucine (Q155L) within the SDL ofintegrindecreased the adhesion ofintegrinto laminin in rat bladder epithelila cell lines.44Allegra and coworkers demonstrated that mutation S47L ofpropeller domain of integrin dramatically reduced the expression level ofintegrin in epithelial cells.45In the current study, flow cytometry analysis demonstrated that ARPE-19 cell expressing S47L andQ155L) mutations significantly affect expression of integrinand subunits on the cell surface and thus interfered with binding to laminin. These data suggested that integrinis, at least in part, involved in RPE cell adhesion to laminin. This observation was consistent with the results by Aisenbrye et al, who
pointed out the importance of integrinin the adhesion of RPE to laminin by demonstrating that ARPE-19 cells could synthesize laminins and adhere them through -containing integrins,and
Several studies reported that integrin1 subunit participated in the adhesion of RPE
cells to Bruch’s membrane.47, 48In this study, we found that surface expression of
-transfectants. The molecular basis for the cross-regulation betweenand1integrin
in-transfectants remained unknown. However, there are several examples of
integrin cross-regulation. Sun et al. have demonstrated that transfection of2 cDNA
into murine mammary carcinoma cell lines result in increased expression of both the and 4 integrin subunits.49
In addition, our results showed the adhesion of -transfectants was better than that of-transfectants in elastic and outer
collagenous layers of Bruch’s membrane explant. Since elastic and outer collagenous
layers are mainly composed of collagen and fibronectin,50which were the ligands of
integrin, we speculated the difference in the behavior between-transfectants and -transfectants in elastic and outer collagenous layers of Bruch’s membrane explant possibly reflected the difference in the expressionintegrin.
In addition to initial attachment, the ability of transplanted RPE cells to proliferate on Bruch’s membrane also has great influence on the success of RPE transplantation. Cell proliferation is known to control by many of the same signaling proteins that play
a role in adhesion and also require a proper interaction of integrin receptors with their
ECM ligands.51, 52Tezel et al., demonstrated that inadequate the number, type or
study, the induction of integrinandoverexpression in -transduced RPE cells led to not only an increase in cells adhesion, but also cell proliferation on deeper
layers of Bruch’s membrane, indicating
The current study has several limitations. First and the major is that porcine Bruch’s
membrane was used for the preparation of different layers of Bruch’s membrane
explants. Although porcine Bruch’s membranes are approximately close to those of
human with typical pentalaminate structure,55they lack age-related alterations in the
molecular composition and ultrastructure. Additional analysis of whether transfection
ofintegrin subunit into ARPE-19 could enhance attachment to human aged
Bruch’s membrane is now undergoing in our laboratory. Second, coherent to Bruch’s
membrane explant model which lack the influence of overlying neurosensory retina
and choroid, the microenvironment of RPE cells seeded onto BM in tissue culture
may be different from the subretinal space. Despite these limitations, the in vivo
Bruch’s membrane explant system is the useful method to quantitatively evaluate the
behavior of various types or genetically modification of human RPE cells on BM
specimens that may resemble the substrate encountered in vivo duration RPE
In conclusion, we have shown that ex vivo gene transfer of integrin genes can
increase the adhesion and proliferation of transplanted RPE cells on Bruch’s
membrane explant. The possibility of ex vivo gene transfer to RPE cell may widen the
scope of this procedure to include gene therapy or adjunctive molecular treatment of
Reference:
1. Klein R, Klein BE, Jensen SC, Meuer SM. The five-year incidence and progression of age-related maculopathy: the Beaver Dam Eye Study. Ophthalmology 1997;104:7-21.
2. West SK. Looking forward to 20/20: a focus on the epidemiology of eye diseases. Epidemiol Rev. 2002;22:64-70.
3. Macular photocoagulation study group. Laser photocoagulation of subfoveal neovascualr lesions in age-related macular degeneration: results of a randomized clinical trial. Arch Ophthalmol. 1991;109:1220-1231.
4. Photodynamic theraoy with verteprofin for age-related macular degeneration. American Acandemy of Ophthalmology. Ophthalmology 2000;107:2314-2317. 5. Krzystolik MG, Afshari MA, Admais AP, et al. Prevention of experimwntal
choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol. 2002;120:338-346.
6. Del Priore LV, Kaplan HJ, Berger AS. Retinal pigment epithelial transplantation in the management of subfoveal choroidal neovascularization. Semin
Ophthalmol. 1997;12:45–55.
7. Del Priore LV, Kaplan HJ, Horbeck R, Jones Z, Swinn M. Retinal pigment epithelial debridement as a model for the pathogenesis and treatment of macular degeneration. Am J Ophthalmol. 1996;122:629–643.
8. Castellarin AA, Nasir MA, Sugino IK, et al. Progressive presumed
choriocapillaris atrophy after surgery for age-related macular degeneration. Retina. 1998;18:143–149.
9. Algvere PV, Berglin L, Gouras P, Sheng Y. Transplantation of fetal retinal pigment epithelium in age-related macular degeneration with subfoveal neovascularization. Graefe’s Arch Clin Exp Ophthalmol. 1994;232:707-716.
10. Binder S, Stolba U, Krebs I, et al. Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration: a pilot study. Am J Ophthalmol. 2002;133:215–225. 11. Algvere PV, Gouras P, Dafgard Kopp E. Long-term outcome of RPE allografts
in non-immunosuppressed patients with AMD. Eur J Ophthalmolo. 1999;9:217-230.
12. Del Priore LV, Kaplan HJ, Tezel TH, et al. Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation. Am J Ophthalmol.
2001;131:472–480.
13. Del Priore LV, Tezel TH, Kaplan HJ. Maculoplasty for age-related macular degeneration: reengineering Bruch’s membrane and human macula. Prog Retin Eye Res 2006;25:539-562.
14. Grossniklaus HE, Hutchinson AK, Capone A, Woolfson J, Lambert HM. Clinicopathologic features of surgically excised choroidal neovascular membranes. Ophthalmology 1994;101:1099-1111.
15. Del Priore LV, Tezel TH, Kaplan HJ. Maculoplasty for age-related macular degeneration: Reengineering Bruch’s membrane and the human macula. Prog. Retinal Eye Res. 2006;25:539-562.
16. Del Priore LV, Tezel TH. Reattachment rate of human retinal pigment epithelium to layers of human Bruch’s membrane. Arch Ophthalmol. 1998;116:335-341.
17. Tezel TH, Del Priore LV, Kaplan HJ. Fate of human retinal pigment epithelial cellsseeded onto layersofhuman Bruch’smembrane.Invest Ophthalmol Vis Sci. 1999;40:467–476.
18. Zarbin MA. Analysis of retinal pigment epithelium integrin expression and adhesion to aged submacular human Bruch’s membrane. Trans Am Ophthalmol Soc 2003;101:499-520.
19. Avery RL, Glaser BM. Inhibition of retinal pigment epithelial cell attachment by a synthetic peptide derived from the cell-binding domain of fibronectin. Arch Ophthalmol 1986;104:1220-1222.
20. Ho TC, Del Priore LV. Reattachment of cultured human retinal pigment epithelium to extracellularmatrix and human Bruch’smembrane.Invest Ophthalmol Vis Sci. 1997;38:1110–1118.
21. Hynes RO. Integrins:bidirectional, allosteric signaling machines. Cell 2002;110:673-687.
22. van der Flier A, Sonnengerg A.Function and interactions of integrins. Cell Tissue Res 2001;305:285-298.
23. Elner SG, Elner VM. The integrin superfamily and the eye. Invest Ophthalmol Vis Sci 1996;37:696-701.
24. Berman AE, Hozlova NI. Integrins: structure and functions. Membr. Cell Biol. 2000;13:207-244.
25. Sonnenberg A. Integrins and their ligands. Curr. Top Microbiol Immunol. 1993;184:7-35.
26. Kikkawa Y, Sanzen N, Fujiwara H, Sonnenberg A, Sekiguchi K. Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by andintegrins. J Cell Sci 2000;113:869-876.
27. Niessen CM, Hogervorst F, Jaspars LH, de Melker AA, Delwel GO, Hulsman EHM, Kuikman I, Sonnenberg A. Theintegrin is a receptor for both laminin and kalinin. Exp Cell Res. 1994;211:360-367.
28. Ebihara N, Watanabe Y, Nakayasu K, Kanai A. The expression of laminin-5 and ultrastructure of the interface between basal cells and underlying stroma in the keratoconus cornea. Jpn J Ophthalmol 2001;45:209-215.
29. Sonnenberg A, Calatat J, Jansen H. et al., Integrincomplex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 1991;113:907-917.
30. Mercurio AM, Rabinovitz I. Towards a mechanistic understanding of tumor invasion-Lessons from the alpha6beta4 integrin. Semin Cancer Biol 2001; 11:129-141.
31. Mainiero F, Murgia C, Wary KK, et al. The coupling ofintegrin to
Ras-MAPK kinase pathways mediated by Shc controls keratocyte proliferation. EMBO J. 1997;16:2365-2375.
32. Mili H, Bellhorn MB, Henkind P. Specializations of the retinochoroidal juncture. Invest Ophthalmol Vis Sci 1975;14:712-717.
33. Marshall GE, Konstas GP, Reid GG, Edwards JG, Lee WR. Type IV collagen and laminin in Bruch’s membrane and basal linear deposit in the human macula. Br. J Ophthalmol 1992;76:607-614.
34. Stepp MA, Michaud SS, Tsidale A, Elwell J, Gipson IK.integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci USA 1990;87:8970-4.
35. Spinardi L, Einheber S, Cullen T, Milner TA, Giancotti FG. A recombinant tail-less integrinsubunit disrupts hemidesomsomes, but does not suppress mediated cell adhesion to laminin. J Cell Biol 1995;129:473-487. 36. Tezel TH, Del Priore LV, Kaplan HJ. Reattachment to a substrate prevents
apoptosis of human retinal pigment epithelium. Graefes Arch Clin Exp Ophthalmol. 1997;235:41-47.
37. Ruzzi L, Gagnous-Palacios L, Pinola M. et al. A homozygous mutation in the integrin6 gene in junctional epidemolysis bullosa with pyloric atresia. J Clin Invest 1997;99:2826-2831.
38. Ashton GHS, Sorelli P, Mellerio JE, Keane FM, Eady RAJ, McGrath JA.64 integrin abnormalities in junctional epidermolysis bullosa with pyloric atresia. Br J Dermatol 2001;144:408-414.
39. van der Neut R, Krimpenfort P, Calafat J, et al. Epithelial detachment due to absence of hemidesmosomes in integrin4 null mice. Nat Genet 1996;13:366-9.
40. Binder S, Stanzel BV, Krebs I, Glittenberg C. Transplantation of the RPE in AMD. Prog. Retinal Eye Res. 2007;26:516-54.
41. Saigo Y, Abe T, Hojo M., Tomita H, Sugano E, Tamai M. Transplantation of transduced retinal pigment epithelium in rats. Invest. Ophthalmol. Vis Sci 2004;45:1996-2004.
42. Lund RD, Adamson P, Sauve Y. et al., subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats. Proc Natl Acad Sci U S A. 2001;14;98:9942-7
43. Takagi J, De Bottis DP, Erickson HP, Springer TA. The role of the specificity-detemining loop of the integrinsubunit I-like domain an autonomous expression, association with thesubunit, and ligand binding. Biochemistry 2002;41:4339-4347.
44. kamata T, Tieu KK, Iric A, Springer TA, Takada Y. Amino acid residues in the apha IIb subunit that are critical for ligand binding to integrin alphaIIbbeta 3 are clustered in the beta-propeller model. J Biol Chem 2001; 276:44275-44283. 45. Tsuruta D, Hopkinson SB, Lane KD, Werner ME, Cryns VL, Jones JCR.
Crucial role of the specificity-determining loop of the integrin4subunit in the
binding of cells to laminin-5 and outside-in signal transduction. J Biol Chem 2003;278:38707-38714.
46. Allergra M, Gagnoux-Palacios L, Gache Y, et al. Rapid decay ofintegrin caused by a mis-sense mutation in the propeller domain results in sever junctional epidermolysis bullosa with pyloric atresia. J. Invest Dermatol 2003;121:1336-1343.
47. Aisenbrey S, Zhang M, Bacher D, Yee J, Brunken WJ, Hunter DD. Retinal pigment epithelial cells synthesize laminins, including laminin 5, and adhere to them through-and 6-containing integrins. Invest Ophthalmol Vis Sci. 2006;47:5537-5544.
48. Chu P, Grunwald GB. Functional inhibition of retinal pigment epithelial cell-substrate adhesion with a monoclonal antibody against the1 subunit of integrin. Invest Ophthalmol Vis Sci 1991;32:1763-1769
49. Chu P, Grunwald GB. Identification of the 2A10 antigen of retinal pigment epithelium as a1 subunit of integrin. Invest Ophthalmol Vis Sci
1991;32:1757-1762.
50. Sun H, Santoro SA, Zutter MM. Downstream events in mammary gland
morphogenesis mediated by reexpression of the alpha2beta1 integrin: the role of the alpha6 and beta4 integrin subunits. Cancer Res. 1998;58:2224-33.
51. Das A, Frank RN, Zhang NL. et al. Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch’s membrane. Arch Ophthalmol 1990;108:421-429.
52. Greenwood JA, Murphy-Ullrich JE. Signaling of de-adhesion in celluar regulation and motility. Microsc Res Tech. 1998;43:420-432.
53. Davey G, Buzzai M, Assoian RK. Reduced expression of alpha5beta1 integin prevents spreading-dependent cell proliferatin. J Cell Sci. 1999;112:4663-4672. 54. Tezel TH, Del Priore LV. Repopulation of different layers of host human
Bruch’smembraneby retinalpigment epithelial cell grafts. Invest Ophthalmol Vis Sci. 1999;40:767–774.
55. Tezel TH, Del Priore LV, Kaplan HJ. Reengineering of aged Bruch’s membrane to enhance retinal pigment epithelium repopulation. Invest Ophthalmol Vis Sci 2004;45:3337-3348.
56. Braekevelt CR. Fine structure of the choriocapillaris, Bruch's membrane and retinal epithelium of the cow. Anat Histol Embryol. 1986;15(3):205-14.
Figure legends
Figure 1. (A). Expression of integrin,andsubunit mRNA in ARPE-19 cells. Integrin mRNA was amplified by RT-PCR using gene-specific primers. PCR products
were elecrophoresed in 1.5% agarose gel and stained with ethidium bromide. Bands
of integrin,andsubunits mRNA were clearly visible in ARPE-19 cells. (B). Flow cytometric analysis of surface-expressed integrinandsubunit in ARPE-19 cells. Histograms depict relative fluorescence intensity (log scale) of negative control
(dot lines) and integrinandsubunit (thick lines).
Figure 2. (A) Flow cytometry analysis of surface-expressed integrinand
subunits in mutant-transfected ARPE-19 cells. Mutatedchain were generated by S ->L substitution at position Ser 47(S47L; mutatedchain were generated by Q ->L substitution at position glutamine 155 ((Q155L)) and R ->W substitution at position arginine 1281(4(R1281W)) . The transfectants were stained with either
an anti-subunit antibody or anti-subunit antibody, and were followed by incubation with an appropriate fluoresceinisothiocyanate (FITC)-conjugated or
phycoerythrin (PE) - conjugated secondary antibody. The cells were analyzed for
as a function of fluorescence intensity (x axis) and are representative of three separate
experiments. Dot lines represent scan obtained with the isotype control antibody; dash
lines represent scan obtained with mock-transfectants; solid line represent
mutant-ormutant-transfectants. (B) Adhesion of ARPE-19 cells expressing6
ormutants on laminin. Cells were seeded on 96-well plates coated with laminin (10g/ml) for 6 hours. Adhesion was measured using a colorimetric assay that measured intracellular dehydrogenase activity. Data represent the mean ± SEM of
triplicate samples from a representative result of three separate experiments. *P<0.05
compared with mock-transfectants and parentalcells, as determined by Student’s t test.
WT=parentalcells
Figure 3. Cell surface expression of the integrinandsubunits incDNA
transfectants andcDNA transfectants (A). Cells were stained with
fluoresceinisothiocyanate (FITC)-conjugated antibodies against these integrins and
analyzed by FACS analysis. The data are expressed as cell number(y axis) plotted as a
function of fluorescence intensity (x axis) and are representative of three separate
experiments. Control was obtained with isotype control antibody. Thin lines
represented scan obtained with untrasfected cells; dash lines represented scan
(B). Adhesion ofcDNA transfectants, cDNA transfectants, mock-transfectants,
parentalcells to fibronectin, vitreonectin, laminin and collagen IV. Cells were seeded
on 96-well plates coated with laminin (10g/ml), fibronectin (10 g/ml), vitreonectin
(10g/ml) and collagen IV (25 g/ml) and after 4 hours of incubation, adhesion was
measured using a colorimetric assay that measured intracellular dehydrogenase
activity. Data represent the mean ± SEM of triplicate samples from a representative
result of three separate experiments. *P<0.05 compared with mock-transfectants and
parentalcells, as determined by Student’s t test. Wild= parentalcells. BSA=bovine
serum albumin; LN=laminin; FN=fibronectin; VN=vitreonectin; Col IV= collagen
type IV.
Figure 4. Reattachment rate (A) and proliferation rate (B) ofcDNA transfectants,
cDNA transfectants, mock-transfectants and parentalcells on different layers of
Bruch’s membrane (BM) explants. For reattachment rate, cells were seeded on
different layers of BM explants and allowed to attach for 24 hours. For proliferation
rate, cells that initial attached were cultured for another 24 hours. Plates were washed
and adhesion was quantified using a colorimetric assay that measured intracellular
dehydrogenase activity. Data represent the mean ± SEM of triplicate samples from a
representative result of three separate experiments. *P<0.05 compared with
parental cells. BL= basal lamina layer; ICL= internal collagenous layer; EL= elastic
layer; ECL= external collagenous layer.
Figure 5. Scanning electron microscopic analysis of cellular morphology and cell
coverage of-transfectants (A, D, G, J),cDNA transfectants (B, E, H, K) and
parental cells (C, F, I, L) seeded to different layers of Bruch’s membrane explants for
7 days. ThecDNA-transfectants could almost completely cover the explant with basal lamina (BL) (A), inner collagenous layer (ICL) (D), elastic layer (EL) (G) and
external collagenous layer (ECL) (J). ThecDNA-transfectants could not survival on EL and ECL. SEM showed that no intact cells were present, revealing the
underlying elastic and collagen fiber of Bruch’s membrane bed (G, H) SEM of
mock-transfectants on BL showed complete coverage, but some small defects were
present. (I). However, mock-transfectants could not be survival on ICL, EL and ECL.
